The overall aim of the proposed research is to gain an understanding of the molecular basis of neuronal differentiation. Nerve growth factor (NGF) induced differentiation of a clonal cell line, PC12, will be used as a model system for neuronal differentiation. The approaches involve the assessment of NGF mediated specific protein phosphorylations as a means of eliciting effects in the target cell which are involved in the expression of the neuronal phenotype. These approaches include (A) the identification of the kinase system responsible for NGF mediated protein phosphorylation. The protein substrate specificity and site of phosphorylation within a protein by NGF-activated kinase will be determined and compared to the cellular kinases activited by EGF, insulin and cAMP. Cellular kinases will also be separated from cell homogenates and their activation in response to NGF measured directly. (B) The general and specific requirements for NGF induced protein phosphorylation in neuronal differentiation will be investigated by (I) specifically activating and blocking cellular kinases to cause or block respectively NGF effects on differentiation and (2) by determining the role of the phosphorylations of tyrosine hydroxylase and vinculin in the NGF induced increase fo catecholamine production and neuronal process extension respectively. These studies are intended to provide a basis for the eventual understanding of the role of protein kinase systems in the growth and development of neurons, the establishment of different developmental pathways of neurons, and the regulation of axonal guidance.